The present invention relates to a novel therapeutic or diagnostic agent containing as an active ingredient at least one nucleic acid and which is particularly useful for the early diagnosis of dysplasias of the stratified squamous epithelium and the cartilage as well as for tumour diagnosis and tumour therapy.
The squamous epithelial carcinoma is the type of oesophageal tumour having the highest incidence. In tumour diagnostics, this tumour type has been classified on the basis of histological criteria into four categories (G1, G2, G3, and G4) wherein the occurrence of histopathological abnormalities is least in G1 tumours and highest in G4 tumours.
Great efforts have been made in medical research to develop suitable methods for tumour therapy. This research has been based on a comprehensive morphological, histological, and molecular tumour diagnosis as well as on the investigation of the generation of tumours. The molecular etiology of tumour generation is not homogenousxe2x80x94however, in a high number of cases mutations (deletions, gene amplifications, translocations of chromosomal portions, etc.) can be detected in the respective tissues which gradually manifest themselves in abnormal differentiation and eventually in uncontrolled cell divisions. In the squamous epithelial carcinomas of the oesophagus, a variety of mutations of oncogenes, tumour suppressor genes, and growth factor genes has been reported (see review in Stemmermann et al., 1994). Interestingly, such mutations were also detected in the tissues adjacent to the tumour which appeared to be histologically normal. It has been assumed that for a tumour to develop the entirety of intracellular damages must exceed a critical threshold value. Therefore, a successful tumour therapy will also depend on its ability to diagnose early stages of tumour development.
From a histopathological point of view, dysplasias of the stratified squamous epithelium are precancerogenic lesions and, thus, may represent potential early stages of tumour development. One of the reasons for the development of dysplasias is the degeneration of the transcriptional control of differentiation-specific genes. Normally, this control is ensured by an orderly co-operation of transcription factors which jointly regulate the activity of these target genes. If it would be possible to show an alteration of already the expression, subcellular localization, or activity, respectively, of these transcription factors, then, these findings might be used as diagnostic markers for an early diagnosis of dysplasias. Regarding squamous epithelial tumours, a useful marker should also be able to indicate an alteration of the expression in malignant tissue.
Pax genes are members of a multigene family containing a conserved DNA sequence called the xe2x80x9cpairedxe2x80x9d box. To date, 9 different Pax genes (Pax1-Pax9) have been isolated from the genomes of humans and mouse (see review in Walther et al., 1991; Stapleton et al., 1993; Wallin et al., 1993). In addition, the xe2x80x9cpairedxe2x80x9d box has also been detected in members of lower classes of animals such as nematodes, drosophila, zebrafish, turtles and chicken (see review in Noll, 1993). The xe2x80x9cpairedxe2x80x9d box codes for the DNA-binding xe2x80x9cpairedxe2x80x9d domain; thus, the proteins encoded by the Pax genes may be assigned to the class of transcription factors (Treisman et al., 1991; Chalepakis et al., 1991; Xu et al., 1995).
Pax genes play an important role in the development of embryonic structures. During the development of the mouse embryo, Pax genes are expressed spatially as well as temporally in specific patterns which partly overlap each other (Gruss and Walther, 1992). The strong instructive effect of the Pax genes during embryogenesis could be demonstrated i.a. by ectopic expression of Pax6 in the imaginal discs of drosophila wings and legs, respectively, using genetic engineering (normally, the Pax6 gene is expressed in the eye primordium). By ectopic Pax6 expression in the imaginal discs of wings or legs, respectively, a nearly complete eye develops in a wrong site (Halder et al., 1995).
By the finding that mutations in Pax genes cause congenital defects in mice but also in men (Pax1: undulated; Pax3: splotch and Waardenburg syndrome; Pax6: small eye and aniridia) the studies performed on this group of genes gained further interest (Balling et al., 1988; Epstein et al., 1991; Tassabehji et al., 1992; Hill et al., 1991; Ton et al., 1991).
The function of the Pax genes is not restricted to embryogenesis. For example, it could be shown that Pax5 protein activates the CD19 gene (CD19 codes for a protein specific for B lymphocytes) (Kozmik et al., 1992), and therefore has also functions in the adult organism. Pax8 is expressed in the thyroid of the adult organism and is involved in the activation of the thyroglobulin and thyroperoxidase genes (Zannini et al., 1992).
Some of the members of the family of Pax genes (Pax1, Pax2, Pax3, Pax6, Pax8) have been identified as proto-oncogenes due to their tumourigenic properties. This identification has been based on transformation tests in which the above-mentioned Pax genes are overexpressed in NIH3T3 cells or 208 cells, respectively, under the control of the cytomegalovirus promoter. Injection of the transformed 208 cells into nude mice caused sarcomas to occur in almost all of the cases (Maulbecker and Gruss, 1993).
The molecular basis for the development of tumours by activation of Pax genes is unknown. It could be shown for a number of rhabdomyosarcomas that as a result of chromosomal translocations Pax3 or Pax7 become fused to the FKHR gene, a transcription factor of the family of forkhead genes. It has been demonstrated that chimeric transcripts of Pax3-FKHR or Pax7-FKHR, respectively, are expressed in rhabdomyosarcomas (Shapiro et al., 1993; Davis et al., 1994).
Evidence for Pax2 expression has been achieved for Wilm""s tumours of the kidney (Dressler and Douglass, 1992). Pax2 is necessary for kidney developmentxe2x80x94its expression, however, is down-regulated as early as during embryogenesis and is no longer detected in normal adult kidney (Dressler et al., 1990).
A therapeutic or diagnostic means is known from DE-A-42 25 569 which contains as an active ingredient at least one nucleic acid hybridizing to a Pax gene. Pax1 through Pax8 are mentioned as Pax genes. Furthermore, the use of such means as a molecular probe in tumour diagnosis and as antisense nucleic acid for the inhibition of gene expression are described. DE-A-42 25 569 does not mention a therapeutic or diagnostic means using a nucleic acid hybridizing to the Pax9 gene nor the uses of such means. This may be due to the fact that at the filing date of DE-A-42 25 569 (03/08/1992), the existence of the Pax9 gene was yet unknown. Further, this German Published Application does only disclose in general the use of such probes in tumour diagnosis or tumour therapy. There are no specific fields of tumour diagnosis or tumour therapy disclosed in which the agent might actually be useful, and the disclosure of which would be of importance considering the variety and heterogeneity of tumours. In particular, this Published Application does not disclose a means which may also be used in the early diagnosis of precancerogenic lesions, particularly of dysplasias of the stratified squamous epithelium.
It is an object of the present invention to provide a novel probe comprising as an active ingredient at least one nucleic acid which is useful for tumour diagnosis and tumour therapy of dysplasias, metaplasias and tumours of epithelial cells and cartilage cells.
According to the invention, this object has been solved by the therapeutic or diagnostic means characterized in more detail in claim 1. Preferred embodiments become clear from the dependent claims and the alternative independent claims.
According to the invention, it has been surprisingly found that a nucleic acid coding for the amino acids corresponding to 30 to 337 of SEQ ID No:2 and hybridizing to the Pax9 gene or a gene derived therefrom may be used in the diagnosis and therapy of dysplasias, metaplasias, and tumours of epithelial cells and cartilage cells. In particular, such nucleic acid is excellently useful in the diagnosis of squamous epithelial carcinomas. A diagnostic means of that type may be of excellent use in the early diagnosis of epithelial dysplasias, metaplasias, and tumours of the stratified squamous epithelium. Using this, for example an early diagnosis of dysplasias as potential precursors of metaplasias and tumours of the respective tissues may be performed at a very early stage in which neither a macroscopical nor a microscopical (!) detection of pathologically altered cells can be performed.
The therapeutic or diagnostic means of the invention contains as an active ingredient at least one nucleic acid comprising (a) a nucleic acid sequence coding for Pax9 protein, (b) a portion thereof, (c) a nucleic acid sequence hybridizing to the nucleic acid sequences of (a) and/or (b) under stringent conditions, or (d) a nucleic acid sequence being complementary to the nucleic acid sequences of (a), (b), and/or (c).
In a further embodiment of the invention, the nucleic acid comprises the sequence coding for amino acids 1-208 and the sequence coding for amino acids 209-341 (see SEQ ID No:2; FIG. 1B.
Due to the cloning technique used, it has been impossible to determine nucleotides 1-4 and 1018-1023, respectively, or the corresponding amino acids 1 and 2 and amino acids 340 and 341, respectively. In the accompanying FIG. 1B, these amino acids have been designated by xe2x80x9c*xe2x80x9d. In contrast, the sequence protocol starts with nucleotide 5 (designated 1) which has actually been sequenced.
The nucleic acid may be a DNA or a RNA which are optionally modified.
Preferably, the nucleic acid of the invention hybridizes to the Pax9 gene under stringent conditions. According to the invention, stringent conditions are defined as conditions allowing for selective and detectable specific binding of the nucleic acid to a Pax9 gene or a derivative thereof, or a Pax9 transcript or a derivative thereof. Preferably, such hybridization under stringent conditions is defined as a hybridization at 42xc2x0 C. in 50% formamide and subsequent washing of the filter at 65xc2x0 C. in an aqueous solution after which still binding of the probe to the Pax9 gene or the Pax9 RNA or derivatives thereof may be detected. If shorter nucleic acids are used as probes, however, it may be necessary to employ less drastic hybridization and/or washing conditions. Highly stringent conditions consist of: washing of the filter in 0.1xc3x97SSC, 0.1% SDS at 68xc2x0 C.
To specifically detect the Pax9 gene, a nucleic acid sequence must be used which is derived from the non-conserved region of the gene and preferably the region not encoding the paired domain. The same applies if the Pax9 gene is to be blocked.
The agent of the invention may be used in an efficient amount as a molecular probe in the diagnosis or therapy of dysplasias, metaplasias, and tumours. Further, it may be used as an antisense nucleic acid for the specific inhibition of Pax9 gene expression.
Furthermore, according to the invention there is provided a method for tumour diagnosis and a method for the inhibition of the Pax9 gene expression employing the agent of the invention in an effective amount.
A further therapeutic or diagnostic means provided according to the invention contains at least one active ingredient in an effective amount comprising at least one Pax9 protein, analogues, portions, conjugates, oligomers, and/or mixtures thereof. Such means will be used in an effective amount in the diagnosis and/or therapy of dysplasias, metaplasias, and tumours of epithelial cells.
In a further embodiment of the invention, there is provided a therapeutic or diagnostic means containing at least one active ingredient comprising at least one antibody against a Pax9 protein, analogues, portions, conjugates, oligomers, and/or mixtures thereof.
The therapeutic and diagnostic means provided according to the invention containing a portion of the Pax9 protein, analogues, conjugates, oligomers, and/or mixtures thereof preferably contains amino acids 132-341, and in a further preferred embodiment contains amino acids 251-341, each shown in FIG. 1B (this method of counting also includes the amino acids not covered by the sequencing technique used; if those are not considered in the counting, one obtains amino acids 130-337 and 249-337, respectively), or amino acids 130-337 or 249-337 according to SEQ ID No:2.
In a further preferred embodiment of the invention, the therapeutic or diagnostic means provided by the invention has at least one active ingredient comprising at least an antibody, preferably a monoclonal antibody against a Pax9 protein, analogues, portions, conjugates, oligomers, and/or mixtures thereof, wherein in specific embodiments of the invention those antibodies may be directed against epitopes localized in regions 132-341 or 251-341, respectively, of the amino acid sequence of FIG. 1B, or 130-337 or 249-337 according to SEQ ID No: 2.
The invention further provides the Pax9 protein with the amino acid sequence according to SEQ ID No: 2, analogues, portions, conjugates, oligomers and/or mixtures thereof.
Nucleotide sequence 1-627 and amino acid sequence 1-208 (SEQ ID No: 2) are already described in Stapleton (1993), and in a specific embodiment, these are not encompassed by the scope of the invention.
The present invention is also directed to a DNA coding for the above-mentioned Pax9 protein, and a RNA derived from said DNA.
The antibodies of the invention are preferably monoclonal antibodies available according to the method of Kxc3x6hler and Milstein in a well-known manner by immunization of a test animal, preferably a mouse, with the Pax9 protein or/and a mixture of Pax9 proteins, preparation of antibody-producing B cells or of spleen cells from the immunized test animal followed by fusion of the antibody-producing cells to a suitable leukemia cell for the generation of hybridomas.
Preferably, the antibodies of the invention may be used in vitro and/or in vivo as means for tumour diagnosis and/or tumour therapy. In this case, the antibodies may also be employed in the form of fragments (e.g. Fab or F(ab)2 fragments) and optionally coupled to a detectable group (enzyme, fluorescent marker, radioactive marker, nuclear resonance marker etc.) or to a toxin (e.g. ricine, diphtheria toxin etc.). The preparation of such antibody derivatives is carried out in a way which is well-known to the skilled artisan in the field of immunology (such as by covalent coupling via a bi-functional linker). The invention also comprises antibodies, in particular monoclonal antibodies directed against a Pax9 protein, analogues, portions, conjugates, oligomers and/or mixtures thereof. These antibodies may be employed in the methods provided according to the invention as well as in the uses claimed.
The therapeutic or diagnostic means provided by the invention are used for diagnosis or therapy of dysplasias, metaplasias, and tumors of epithelial cells, preferably of the stratified squamous epithelium, and of cartilage cells. The diagnosis and therapy of alterations in oesophagus, skin, such as psoriasis, buccal mucosa, tongue, cornea, vagina, cervix, endometrium, anus, sebaceous glands of the skin, and cartilage represent preferred fields of the invention. In particular, a potential field of the present invention is the early diagnosis of epithelial cell dysplasias.
Diagnosis and in particular early diagnosis, as well as therapy in the case of metaplasias is an important field of the present invention. A metaplasia, and thus, in this case the conversion of an epithelium into a stratified squamous epithelium may theoretically occur at every site of the body where xe2x80x9cnon-squamous epitheliaxe2x80x9d are present. It is for example well-known that the simple squamous epithelium of the trachea of smokers is frequently converted into a stratified squamous epithelium. Another important example is the conversion of the endometrial epithelium (generally a monolayer) into a stratified squamous epithelium. These sites obviously show an increased risk for the development of a squamous epithelial carcinoma. The stratified squamous epithelia can be diagnosed and treated by the agent provided by the invention which contains at least one active ingredient having at least one nucleic acid hybridizing to a Pax9 gene or a derivative thereof.
As detailed in the following, it could be demonstrated according to the invention that higher amounts of the Pax9 protein are localized in the cytoplasm in epithelial cell dysplasias than in normal cells. Therefore, according to the invention, recombinant vectors may be used having a sequence coding for a Pax9 protein, an analogue, portions, conjugates, oligomers, and mixtures thereof, being in a preferred embodiment operably linked to one or more signal sequences for the directed transport of the Pax9 protein into the cell nucleus. This vector may for example be derived from a plasmid or a viral vector. Typical vectors well-known or still to be developed in the field of gene therapy may be used according to the invention. Preferably, this vector is an expression vector which may be expressed in procaryotic as well as in eucaryotic cells.
The present invention also comprises eucaryotic cells and procaryotic cells transformed by a vector containing the Pax9 gene of the invention.
The vectors containing the Pax9 gene according to the invention are useful in somatic gene therapy of humans. For example, they may be used for terminal differentiation of tumour cells and tumour precursor cells.
Also comprised by the invention are analytic kits for the analysis of Pax9 expression in cartilage as well as epithelial cells, and in particular in cells of the squamous epithelium with respect to dysplasias, metaplasias, and tumours.